PROJECT SUMMARY/ABSTRACT The causality of human environmental exposures and disease development is irrefutable. Human cells deploy highly coordinated and structured mechanisms in response to ubiquitous environmentally related genotoxic damage. Amongst these, versatile nucleotide excision repair (NER) efficiently mends helix-distorting lesions by global genomic repair (GGR) and transcription-coupled repair (TCR) sub-pathways. Ongoing work of past project years has provided in-depth mechanistic insights about the individual steps of GGR and TCR as well as the crucial elements of their regulation. This continuation grant application will extend the scope of these studies along a similar overall theme to demonstrate the role of ubiquitination/deubiquitination during the initial damage recognition phase and following the successful completion of repair during the restoration of intact chromatin. Overall work is based on the premise that specific ubiquitinating/ deubiquitinating activities orchestrate the coordinated targeting of core repair and regulatory factors to damage sites. As the final aftermath of damage response, the deubiquitination events herald the cell cycle progression and transcription recovery. Thus, the proposed studies plan to dissect the dynamics of DNA damage recognition and its elaborate processing against the regulatory backdrop of specific ubiquitination/ deubiquitination of key DNA repair proteins. Four inter-related specific aims are proposed to, (1) reveal the cellular components operating in deubiquitination of crucial NER recognition factor XPC during GGR; (2) establish the underlying mechanism by which Cul4A-DDB1-CSA E3 ubiquitin ligase complex mediates the regulation of TCR; (3) delineate the molecular machinery operational in incorporating the mono- ubiquitinated histone 2A (uH2A) within repaired genomic sites; and (4) distinguish the role of putative deubiquitinase and functional relevance of uH2A deubiquitination in restoring chromatin. Powerful in situ technology and related resources established in the previous project years will be applied to experimentally dissect the molecular/cellular components of ubiquitination/deubiquitination via spatiotemporal in vivo assembly/disassembly of repair and regulatory factors, in vivo and in vitro DNA-protein/protein-protein interactions and kinetics of lesion removal in conjunction with the ultimate fate of ubiquitinated factors. Identification, function and regulation of these critical interacting factors and repair pathway events influencing the overall genomic stability, so fundamental to normal cellular existence, has obvious significance in understanding the cause and treating human environment based diseases.